Circular dichroic evidence for an ordered sequence of ligand/binding site interactions in the catalytic reaction of the cAMP-dependent protein kinase.
Circular dichroic evidence for an ordered sequence of ligand/binding site interactions in the catalytic reaction of the cAMP-dependent protein kinase.
复制标题
cAMP 依赖性蛋白激酶催化反应中配体/结合位点相互作用的有序序列的圆二向色证据。
DOI：
10.1021/bi00333a024
复制
发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Walsh,DA
中科院分区：
文献类型：
--
作者：
Reed,J;Kinzel,V;Kemp,BE;Cheng,HC;Walsh,DA
Institute of Experimental Pathology, GermanCancer Research Center, D-6900 Heidelberg, Federal Republic of Germany, Department of Medicine, University of Melbourne, Melbourne, Australia, and Department of Biological Chemistry, School of Medicine, University of California, Davis, California 95616 Received August 6, 1984 abstract: A limiting requirement for substrate specificity of the cAMP-dependent protein kinase is the presence of one or two basic residues located to the N-terminal side of the target substrate serine. Furthermore, circular dichroic (CD) studies have shown that binding of protein substrateinvolves a series of at least two independent conformational changes in the enzyme, each of which is initiated by a recognition signal on the substrate protein. The present study attempts to elucidate further the complete sequence of enzyme/ligand interactions by using the synthetic substrate peptide Kemptide and analogues differing from it at crucial points in the sequence: the Ala-peptide, where alanine is substituted for the target serine, andD-Ser-Kemptide, where the target serine is in the D rather than the L configuration. Examination of the effects of binding of these substrates on the intrinsic UV CD of the enzyme and the induced CD inthe presence of Blue Dextran has revealed a third step in the substrate/enzyme binding interaction. Although sections of the conformational change at the active site are dependent on the basic subsite and the serine hydroxyl group on the peptide, respectively, the complete conformational change requires that the substrate be bound in random coil conformation. Where this does not occur, the kinetics show that the peptide will not act either as substrate or as inhibitor of the enzyme. Further, the interaction between the serine hydroxyl group and an enzyme tyrosine residue, previously observed, appears to be dependent on the correct orientation as well as the mere presence of the target-OH group. Taken together, the data allow one to construct an ordered sequence of conformational changes taking place upon substrate binding leadingto the final form of the active site.
